Thymineless Death Lives On: New Insights into a Classic Phenomenon

Khodursky, Arkady B.; Guzmán, Elena C.; Hanawalt, Philip C.

doi:10.1146/annurev-micro-092412-155749

Cited by 49 publications

(60 citation statements)

References 124 publications

(124 reference statements)

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“…When E. coli cells are starved of the DNA precursor dTTP, they quickly lose viability, a phenomenon named thymineless death (TLD) (Barner & Cohen 1954). Although TLD is highly conserved from bacterial to human cells and has been exploited by popular anticancer and bactericidal drugs such as 5-fluorouracil and trimethoprim, respectively, the precise mechanism of cell lethality in TLD is unknown (Guzm an & Mart ın 2015; Khodursky et al 2015). Several groups have recently reported that approximately 600-700 kb of genomic DNA centered around oriC is lost during TLD in E. coli, which is likely to contribute to lethality in TLD (Fonville et al 2010;Sangurdekar et al 2010;Kuong & Kuzminov 2012).…”

Section: Discussionmentioning

confidence: 99%

Replication fork progression is paused in two large chromosomal zones flanking the DNA replication origin in Escherichia coli

et al. 2016

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Although the speed of nascent DNA synthesis at individual replication forks is relatively uniform in bacterial cells, the dynamics of replication fork progression on the chromosome are hampered by a variety of natural impediments. Genome replication dynamics can be directly measured from an exponentially growing cell population by sequencing newly synthesized DNA strands that were specifically pulse-labeled with the thymidine analogue 5-bromo-2 0 -deoxyuridine (BrdU). However, a short pulse labeling with BrdU is impracticable for bacteria because of poor incorporation of BrdU into the cells, and thus, the genomewide dynamics of bacterial DNA replication remain undetermined. Using a new thymidine-requiring Escherichia coli strain, eCOMB, and high-throughput sequencing, we succeeded in determining the genomewide replication profile in bacterial cells. We also found that fork progression is paused in two~200-kb chromosomal zones that flank the replication origin in the growing cells. This origin-proximal obstruction to fork progression was overcome by an increased thymidine concentration in the culture medium and enhanced by inhibition of transcription. These indicate that DNA replication near the origin is sensitive to the impediments to fork progression, namely a scarcity of the DNA precursor deoxythymidine triphosphate and probable conflicts between replication and transcription machineries.

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Section: Discussionmentioning

confidence: 99%

Replication fork progression is paused in two large chromosomal zones flanking the DNA replication origin in Escherichia coli

et al. 2016

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“…By doing so, the synthesis of the pyrimidine thymidine is stalled, which is an essential nucleoside required for DNA replication (Singh et al, 2015). 5-Fluorouracil causes a drop on dTMP and then cells undergo cell death via thymineless death (Khodursky et al, 2015;Singh et al, 2015).…”

Section: Methodsmentioning

confidence: 99%

Antimicrobial peptide induced-stress rendersStaphylococcus aureussusceptible to toxic nucleoside analogues

Rodríguez‐Rojas¹,

Nath²,

Be³

et al. 2020

Preprint

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Cationic antimicrobial peptides (AMPs) are active immune effectors of multicellular organisms and also considered as new antimicrobial drug candidates. One of the problems encountered when developing AMPs as drugs is the difficulty to reach sufficient killing concentrations under physiological conditions. Here, using pexiganan, a cationic peptide derived from a host defence peptide of the African clawed frog and the first AMP developed into a drug, we studied if sub-lethal effects of AMPs can be harnessed to devise treatment combinations. We studied the pexiganan stress response of Staphylococcus aureus at sub-lethal concentrations using a whole proteomics approach by liquid chromatography coupled with mass spectrometry. Several proteins involved in nucleotide metabolism were elevated, suggesting a metabolic demand. We then show that S. aureus is highly susceptible to antimetabolite nucleoside analogues when exposed to pexiganan, even at sub-inhibitory concentrations. These findings could be used to enhance pexiganan potency while decreasing the risk of resistance emergence, and our findings can likely be extended to other antimicrobial peptides.2

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“…As previously mentioned, inhibition of de novo synthesis of dTTP triggers thymineless death phenomena (Ahmad et al, 1998;Khodursky et al, 2015). In T. brucei the de novo synthesis of dTMP is catalyzed by the bifunctional enzyme dihydrofolate reductase-thymidylate synthase (DHFR-TS) which is essential for parasite survival in the absence of dThd (Sienkiewicz et al, 2008).…”

Section: Tk-depleted Cells Exhibit Reduced Dttp Levels and Accumulatementioning

confidence: 99%

Cell cycle regulation and novel structural features of thymidine kinase, an essential enzyme in Trypanosoma brucei

Valente

Timm

Castillo-Acosta

et al. 2016

Molecular Microbiology

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Thymidine kinase (TK) is a key enzyme in the pyrimidine salvage pathway which catalyzes the transfer of the γ-phosphate of ATP to 2'-deoxythymidine (dThd) forming thymidine monophosphate (dTMP). Unlike other type II TKs, the Trypanosoma brucei enzyme (TbTK) is a tandem protein with two TK homolog domains of which only the C-terminal one is active. In this study, we establish that TbTK is essential for parasite viability and cell cycle progression, independently of extracellular pyrimidine concentrations. We show that expression of TbTK is cell cycle regulated and that depletion of TbTK leads to strongly diminished dTTP pools and DNA damage indicating intracellular dThd to be an essential intermediate metabolite for the synthesis of thymine-derived nucleotides. In addition, we report the X-ray structure of the catalytically active domain of TbTK in complex with dThd and dTMP at resolutions up to 2.2 Å. In spite of the high conservation of the active site residues, the structures reveal a widened active site cavity near the nucleobase moiety compared to the human enzyme. Our findings strongly support TbTK as a crucial enzyme in dTTP homeostasis and identify structural differences within the active site that could be exploited in the process of rational drug design.

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Thymineless Death Lives On: New Insights into a Classic Phenomenon

Cited by 49 publications

References 124 publications

Replication fork progression is paused in two large chromosomal zones flanking the DNA replication origin in Escherichia coli

Replication fork progression is paused in two large chromosomal zones flanking the DNA replication origin in Escherichia coli

Antimicrobial peptide induced-stress rendersStaphylococcus aureussusceptible to toxic nucleoside analogues

Cell cycle regulation and novel structural features of thymidine kinase, an essential enzyme in Trypanosoma brucei

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